Sensitizing effect of the phosphatidylinositol 3-kinase inhibitor wortmannin on thermal neutron irradiation with or without boron compound

Wortmannin, a phosphatidylinositol 3-kinase inhibitor, has been found to be an efficient radiosensitizer. We have investigated the radiosensitizing effect of wortmannin on cell killing against thermal neutrons produced by the Kyoto University Research (KUR) reactor. Wortmannin was added to cells 2 hours before irradiation and removed 16 hours after irradiation. Cells were irradiated by thermal neutrons with or without boron at 0, 10, and 20 ppm. The biological end point of cell survival was measured by colony formation assay. The D0 values of thermal neutrons in different boron concentrations, 0, 10, and 20 ppm were 1.2, 1.1, and 1.0 Gy, respectively. When cells were treated with wortmannin, the D0 values decreased to 0.5, 0.6, and 0.8 Gy at boron concentrations of 0, 10, and 20 ppm, respectively. Wortmannin enhanced cell death against thermal neutron irradiation especially in the absence of boron. Thus, our results suggest that wortmannin may be useful to combine with boron neutron capture therapy (BNCT) treatment when boron uptake by cells is limited.     

The biological effect of the phosphatidylinositol 3-kinase wortmannin following thermal neutron irradiation

PURPOSE: Wortmannin, a phosphatidylinositol 3-kinase inhibitor, has been found to inhibit DNA-dependent protein kinase (DNA-PKcs) and to be an efficient radiosensitizer. We investigated the radiosensitizing effect of wortmannin on cell killing by thermal neutrons and the occurrence of mutations at the HPRT locus of CHO cells. MATERIALS AND METHODS: Wortmannin was added to cells at a final concentration of 20 microM two hours before irradiation. Cells were irradiated with thermal neutrons. The biological end point of cell survival was measured by colony formation assay. Mutagenicity was calculated from the mutation frequency at the HPRT locus. RESULTS: Wortmannin promoted cell death following thermal neutron irradiation especially in the absence of boron. However, mutagenicity was decreased by the wortmannin treatment. CONCLUSION: Our results suggest that the inhibition of DNA-PKcs by wortmannin would be an effective radiosensitizer for BNCT and might bring about a reduction in the frequency of mutation by blocking the mis-joining of DNA damage during the repair process.     

Low dose of gamma irradiation enhanced boronophenylalanine uptake in head and neck carcinoma cells for boron neutron capture therapy

This study attempted to increase the boron uptake of human head and neck carcinoma SAS cells for BNCT by using a gamma dose of 0.1 Gy for combined treatment. Intracellular boron concentrations in 25 μg B/mL medium of BPA treated and BPA combined gamma-irradiation treated SAS cells were 73.8±1.73 and 95.15±1.36 ppm, respectively. After neutron irradiation, the G2/M-phase cell populations of untreated, BPA treated and BPA combined gamma-irradiation treated SAS cells were 19.31±1.71%, 52.47±2.25% and 59.19±2.63%, respectively. Experimental results indicate that the low dose gamma radiation with combination BPA treatment has the highest killing rate after neutron irradiation. Capable of significantly increasing the G2/M arrest after neutron irradiation, the combined treatment of a low dose of gamma irradiation with 25 μg B/mL medium of BPA also provided a higher killing effect for BNCT.     

The radiobiology of 24 keV neutrons. Measurement of the relative biological effect free-in-air, survival and cytogenetic analysis of the biological effect at various depths in a polyethylene phantom and modification of the depth-dose profile by boron 10 for V79 Chinese hamster and HeLa cells

HeLa human carcinoma cells and V79 Chinese hamster fibroblasts have been irradiated in vitro with a beam of neutrons with a nearly pure 24 keV spectrum. The relative biological effectiveness (RBE) of the filtered neutron beam relative to 60Co gamma radiation was determined by irradiation of cell cultures "free-in-air". The values obtained for the RBE at 37% survival were 5.8 +/- 0.8, at a dose of 0.69 +/- 0.06 Gy for the HeLa cells and 3.14 +/- 1.1 at a dose of 1.09 +/- 0.091 Gy for the V79 cells. Cytogenetic analysis of the damage in irradiated V79 cells gave an RBE of 6.7 +/- 1.4. Irradiation in a polyethylene phantom markedly attenuated the beam's biological effect. For both cell lines 2 cm of polyethylene virtually eliminated cell killing. Addition of boron 10 to the medium led to increased cell killing and a value of 4 was obtained for the RBE of the 10B(n, alpha)7Li reaction in HeLa cells.     

Augmentation of wound healing by ascorbic acid treatment in mice exposed to gamma-radiation

PURPOSE: Because of the crucial practical importance of acute radiation exposure associated with combined injuries, the study was undertaken to investigate the effect of various doses of ascorbic acid on the survival and healing of wounds in mice exposed to whole-body gamma-radiation. MATERIALS AND METHODS: Animals were given double-distilled water or different doses of ascorbic acid by intraperitoneal injection before exposure to 0 or 10 Gy whole-body gamma-radiation to evaluate the effect of ascorbic acid on radiation-induced mortality. The animals were monitored daily for the symptoms of radiation sickness and mortality. In a separate experiment, animals were administered with either double-distilled water or different doses of ascorbic acid before exposure to 0 or 6 Gy whole-body gamma-radiation to investigate the effect of ascorbic acid on the irradiated wound. A full-thickness skin wound was created on the dorsum of the irradiated mice and the progression of wound contraction was monitored by capturing video images of the wound at various post-irradiation periods. RESULT: Treatment of mice with various doses of ascorbic acid elevated survival of mice and a highest number of survivors (67 and 33% for 10 and 30 days post-irradiation) was observed for 250 mg kg(-1) (p<0.002 and<0.02 for 10- and 30-day survival, respectively). Ascorbic acid treatment caused a dose-dependent elevation in the wound contraction and highest contraction was observed for 250 mg kg(-1). The wound contraction was significantly greater at 3 (p<0.005), 6 (<0.05) and 9 (<0.05) days post-irradiation with 250 mg kg(-1) ascorbic acid. The complete healing of the wound was effected by day 22.8 post-irradiation in the ascorbic acid-treated irradiation group. CONCLUSION: Administration of ascorbic acid protected mice against radiation-induced sickness, mortality and improved healing of wounds after exposure to whole-body gamma-radiation. Additional studies will be directed toward analysing the role of successive administration of ascorbic acid to protect non-target tissues during radiotherapy and in initiating and supporting the cascade of tissue repair processes in radiotherapy delayed wounds.     

辐射诱发淋巴细胞凋亡生成与抑制作用研究

研究了辐射诱发的人外周血淋巴细胞凋亡生成，以及水溶性维生素Ｅ类似物－Ｔｒｏｌｏｘ对辐射诱导人外周血淋巴细胞凋亡的抑制作用。照后３０分钟内Ｔｒｏｌｏｘ能有效地阻抑ＤＮＡ片段形成，而在照前或受照中加入Ｔｒｏｌｏｘ均不能抑制ＤＮＡ片段形成，揭示Ｔｒｏｌｏｘ并不是通过清除照射过程中产生的自由基而起作用。照后３０分钟内加Ｔｒｏｌｏｘ，２小时后撤去，同样能抑制ＤＮＡ片段形成，表明Ｔｒｏｌｏｘ能不可逆地阻抑细胞凋亡早期的＂关键＂事件。     

EGF-receptor targeted liposomes with boronated acridine: growth inhibition of cultured glioma cells after neutron irradiation

PURPOSE: To study survival of cultured U-343MGaCl 2:6 glioma cells after incubation with boron-containing liposomes targeting the epidermal growth factor receptor following neutron irradiation. MATERIALS AND METHODS: Epidermal growth factor-tagged liposomes were loaded with water-soluble boronated acridine developed for boron neutron capture therapy, (BNCT). Cellular uptake and distribution were studied. Further, cells were placed at 3 cm depth in a phantom and exposed to an epithermal neutron beam to study clonogenic cell survival. RESULTS: The cellular uptake of boron reached 90 ppm and it was determined by subcellular fractionation that most of the cell-associated boron was located outside of the nucleus. For clonogenic survival, the cells were incubated with epidermal growth factor receptor-targeted liposomes for 4 hours resulting in a cellular concentration of 55 ppm boron (11 ppm 10B). At a fluence of 3 x 10(12) neutrons/cm2 the cell killing effect of the boron-containing epidermal growth factor-liposomes was about ten times higher than for neutrons only. Furthermore, theoretical calculation of the survival by enriched compound (55 ppm 10B), using the parameters from non-enriched compound (11 ppm 10B), shows that the killing effect in this case would be approximately five orders of magnitude higher than for neutrons only. CONCLUSION: The results in this study show that epidermal growth factor-receptor targeted liposomes are suitable as tumor-cell delivery agents of boron for BNCT and support further studies to demonstrate their effectiveness in vivo.     

Post-irradiation Defects in Newly Synthesized DNA: Neutrons and Electrons Compared and the Effect of Misonidazole

The role of cellular membranes in thymocyte apoptosis has been examined. Trolox, a water soluble analogue of vitamin E and inhibitor of membrane damage, inhibits DNA fragmentative in thymocytes exposed to γ-radiation. Trolox is most effective in inhibiting DNA fragmentation when added to cells within 30 min post-irradiation. Exposure to trolox only during irradiation did not prevent DNA fragmentation, suggesting that it does not work by scavenging free radicals generated during radiation exposure. Incubation of the irradiated cell suspension with trolox for 2 h post-irradiation was sufficient to prevent DNA fragmentation measured at 24 h in irradiated cells. This suggests that trolox irreversibly inhibits a cellular lesion required for apoptosis. The induction of DNA fragmentation appears to be related to a concurrent, pronounced flow of Ca²⁺ into the cell. At 3 h post-irradiation the amount of Ca²⁺ in irradiated thymocytes was more than twice that of unirradiated thymocytes. Membrane damage has been shown to affect the transport of Ca²⁺. Trolox treatment completely blocked the radiation-induced influx of Ca²⁺ into the thymocytes. These results suggest that membrane damage is a critical lesion that is involved in DNA fragmentation in thymocyte apoptosis.     

A basic study on Hypericin-PDT in vitro

The effect of photo dynamic therapy (PDT) using hypericin as a photosensitiser and the effect of PDT on intracellular ATP levels using different lamps in a human leukemic monocyte lymphoma cell line (U937) were studied. The time required for hypericin to penetrate into the cancer cells was 1h, and incubation for more than 3h post-irradiation with hypericin-PDT was required to observe effects. Thus, if cancer cell death does not occur immediately following irradiation, it is unnecessary to perform additional irradiation, as most of the cells die via apoptosis during the incubation period post-irradiation. When hypericin-PDT was performed using a Na-Li lamp as a light source, the cell viability decreased approximately 55% immediately following irradiation for 5min; however, after a 5-h post-irradiation incubation, the cell viability approached 0%. Concurrently, intracellular ATP levels increased markedly; thus, irradiation (0.225J/cm(2)) for 5min provided the best results in terms of the highest degree of cancer cell apoptosis. Similar experiments were performed using three different LED lamps respectively. When cells were treated with the LED lamps, with maximum peaks of 599nm and 595nm, the cell viability approached 0% after incubation for 5h following 15min of irradiation (0.04J/cm(2) and 0.099J/cm(2), respectively). We confirmed that incubating the cells for more than 3h in a 100× diluted hypericin solution was the most effective for PDT and that a LED lamp of low light intensity led to the highest apoptosis rate in the U937 cells.     

重组人白细胞介素-11和姜黄素对中子照射致小鼠肠道损伤的治疗作用

目的 探讨重组人白细胞介素-11(rhIL-11)和姜黄素对中子照射致小鼠肠道损伤的治疗作用。方法 选取二级雄性BALB/c小鼠140只,随机分为健康对照组(20只)、单纯照射组(60只)、IL-11治疗组(30只)和姜黄素治疗组(30只)。单纯照射组小鼠采用3 Gy中子全身均匀照射,两个治疗组于照射后即日分别腹腔注射500μg·kg^-1·d^-1的rhIL-11或200 mg·kg^-1·d^-1的姜黄素,每天1次,连续给药5d。观察各组动物的死亡率。照射后6 h、1、3和5 d分别处死健康对照组和单纯照射组动物,3和5 d分别处死两个治疗组动物。取空肠标本,用HE深色、核仁组成区嗜银蛋白染色、Feulgen氏染色和图像死亡分析等,检测空肠病理形态变化、空肠上皮细胞核内嗜银蛋白含量和DNA含量变化。结果 照后5d,单纯照射组小鼠全部死亡,rhIL-11治疗组存活剩余2只,姜黄素治疗组存活剩余3只。3Gy中子照射后6 h～3 d内,空肠黏膜大面积坏死脱落,3d偶见隐窝细胞再生,5d见较多新生隐窝;IL-11治疗组在照后3d隐窝再生较明显,5d见大量新生绒毛;姜黄素治疗组在照后3d隐窝有部分再生,5d可见新生绒毛,排列结构较IL-11治疗组紊乱,绒毛高度较IL-11治疗组略低。照后5d,两个治疗组小鼠空肠上皮细胞AgNOR含量和DNA含量均高于单纯照射组(F＝0.015～0.035,P<0.05)。结论 3 Gy中子照射引起小鼠空肠明显损伤,rhIL-11和姜黄素治疗可减轻损伤并促进肠道上皮再生修复,对中子辐射肠上皮损伤具有保护作用。     

Correlation between non-repairable DNA lesions and fixation of cell damage by hypertonic solutions in Chinese hamster cells

In Chinese hamster HA-1 cells, killing induced by gamma-rays was enhanced by post-irradiation treatment with hypertonic solution (0.5 mol/l NaCl in phosphate buffered saline, pH 7.2) for 20 min. The initial DNA double-strand breaks (dsb) induced by gamma-rays were repaired during post-irradiation treatment with hypertonic solution. However, hypertonic treatment following gamma-irradiation enhanced the frequency of non-repairable dsb, as compared with the frequency after incubation at 37 degrees C following gamma-irradiation. Hypertonic treatment did not affect the initial half-time for rejoining of dsb. Hypertonic treatment did not enhance cell killing, nor did it enhance the non-repairable dsb when the irradiated cells were incubated at 37 degrees C for 2 h. These results suggest that fixation of gamma-ray-induced potentially lethal damage by hypertonic treatment results from inhibition of the rejoining of dsb.     

Effect of free radicals induced by ultrasonic cavitation on cell killing

The present study was undertaken to elucidate the mechanism of in vitro cell killing induced by 1.0 MHz continuous wave ultrasound at an intensity of 5.8 W/cm2. The chemical effects and mechanical effects arising from acoustic cavitation were determined by the amount of liberated iodine and the number of DNA double-strand breaks, respectively. The survival of mouse L cells immediately after irradiation was estimated by counting the number of cells which are not stained by trypan blue and the clonogenicity of surviving cells remaining immediately after irradiation was monitored by colony-forming ability. The effectiveness of the dissolved gases in liberating iodine was in the order O2 greater than Ar greater than N2 greater than N2O approximately 0. However, the effect of dissolved gases on the yield of double-strand breaks of DNA and on the two kinds of end points of cell killing was in the order O2 = Ar = N2 greater than N2O approximately 0. These results suggest that the different amounts of free radicals induced by ultrasound are not directly related to the ultrasonically induced cell killing. The presence of cysteamine (2 mmol dm-3) during sonication completely inhibited a decrease in clonogenicity of surviving cells, but did not inhibit that of cell survival immediately after sonication. These results suggest that the decrease of survival immediately after sonication is due to mechanical shear stress arising from cavitation, while the decrease of clonogenicity of the remaining surviving cells is due to free radicals induced by cavitation. The contribution of free radicals to total cell killing was estimated as about 1 per cent at the level of 95 per cent cell killing immediately after sonication.     

EGF-receptor targeted liposomes with boronated acridine: Growth inhibition of cultured glioma cells after neutron irradiation

Purpose: To study survival of cultured U-343MGaCl 2:6 glioma cells after incubation with boron-containing liposomes targeting the epidermal growth factor receptor following neutron irradiation.

Materials and methods: Epidermal growth factor-tagged liposomes were loaded with water-soluble boronated acridine developed for boron neutron capture therapy, (BNCT). Cellular uptake and distribution were studied. Further, cells were placed at 3 cm depth in a phantom and exposed to an epithermal neutron beam to study clonogenic cell survival.

Results: The cellular uptake of boron reached 90 ppm and it was determined by subcellular fractionation that most of the cell-associated boron was located outside of the nucleus. For clonogenic survival, the cells were incubated with epidermal growth factor receptor-targeted liposomes for 4 hours resulting in a cellular concentration of 55 ppm boron (11 ppm ¹⁰B). At a fluence of 3 × 10¹² neutrons/cm² the cell killing effect of the boron-containing epidermal growth factor-liposomes was about ten times higher than for neutrons only. Furthermore, theoretical calculation of the survival by enriched compound (55 ppm ¹⁰B), using the parameters from non-enriched compound (11 ppm ¹⁰B), shows that the killing effect in this case would be approximately five orders of magnitude higher than for neutrons only.

Conclusion: The results in this study show that epidermal growth factor-receptor targeted liposomes are suitable as tumor-cell delivery agents of boron for BNCT and support further studies to demonstrate their effectiveness in vivo.     

Gadolinium neutron capture in glioblastoma multiforme cells

Purpose: A proof of principle for cell killing by Gadolinium (Gd) neutron capture in Magnevist® preloaded Glioblastoma multiforme (GBM) cells is provided.

Materials and methods: U87cells were pre-loaded with 5 mg/ml Magnevist® (Gd containing compound) and irradiated using an enhanced neutron beam developed at NIU Institute for Neutron Therapy at Fermilab. These experiments were possible because of an enhanced fast neutron therapy assembly designed to use the fast neutron beam at Fermilab to deliver a neutron beam containing a greater fraction of thermal neutrons and because of the development of improved calculations for dose for the enhanced neutron beam. Clonogenic response was determined.

Results: U87 cell survival after γ irradiation, fast neutron irradiation and irradiation with the enhanced neutron beam in the presence or absence of Magnevist® were determined.

Conclusions: U87 cells were the least sensitive to γ radiation, and increasingly sensitive to fast neutron irradiation, irradiation with the enhanced neutron beam and finally a significant enhancement in cell killing was observed for U87 cells preloaded with Magnevist®. The sensitivity of U87 cells pre-loaded with Magnevist® and then irradiated with the enhanced neutron beam can at least in part be attributed to the Auger electrons emitted by the neutron capture event.     

Characterization of ultra high energy neutron beam generated by 500 MeV proton beam

An ultra high energy neutron facility was constructed at PARMS, University of Tsukuba, to produce a neutron beam superior to an X-ray beam generated by a modern linac in terms of dose distribution. This has been achieved using the reaction on a thick uranium target struck by 500 MeV proton beam from the booster-synchrotron of High Energy Physics Laboratory. The percentage depth dose of this neutron beam is nearly equivalent to that of X-rays at around 20 MV and the dose rate of 15 cGy per minute. Relative biological effectiveness of this neutron beam has been estimated on the cell killing effect by the use of HMV-I cell line. Resultant survival curve of cells after the neutron irradiation shows the shoulder with n and Dq of 8 and 2.3 Gy, respectively. RBE value at 10(-2) survival level for the present neutron, compared with 137Cs gamma-rays is 1.24. The result suggests that the biological effects of high energy neutrons are not practically large enough whenever the depth dose distribution of neutrons becomes superior to high energy linac X-rays.     

12C重离子束对人淋巴细胞增殖、周期和凋亡的影响

目的 探讨¹²C重离子束对人淋巴细胞增殖以及周期、凋亡的影响.方法 ¹²C重离子束照射人淋巴细胞Peng-EBV,吸收剂量分别为0(对照组)、0.5、2.0 Gy.照射后用MTS法检测细胞增殖活力,流式细胞仪检测细胞周期和细胞凋亡.结果 与对照组相比,0.5 Gy照射可以增加细胞增殖活力(t=2.66~14.45, P<0.05),而2.0 Gy照射降低了细胞活力(t=7.65~64.45, P<0.05).受照射细胞的活力存在一个恢复和下降过程,受照后48 h内,细胞数量呈增加趋势,但72 h时细胞数量下降.照射后48 h,两组细胞G₂/M期呈明显上升趋势,高于对照组(t=2.01~99.80,P<0.05),且2.0 Gy组的周期阻滞较0.5 Gy组严重;照射后30 d,细胞周期阻滞恢复到正常水平.照射后12、24、48 h,两照射组与对照组相比,细胞凋亡率差异有统计学意义(t=-3.05~-1.05,P<0.05),在照后24 h最高,48 h明显下降,30 d恢复到对照水平.结论 ¹²C离子束辐射影响人淋巴细胞增殖,诱导人淋巴细胞发生明显的G₂/M期阻滞,并且明显地促进细胞凋亡.     

Treatment of mice with stem bark extract of Aphanamixis polystachya reduces radiation-induced chromosome damage

PURPOSE: Normal tissue radiosensitivity is the major limiting factor in radiotherapy of cancer. The use of phytochemicals may reduce the adverse effects of radiation in normal tissue. The effect of ethyl acetate fraction of Aphanamixis polystachya (EAP) was investigated on the radiation-induced chromosome damage in the bone marrow cells of Swiss albino mice exposed to various doses of gamma-radiation. MATERIALS AND METHODS: The mice were divided into two groups, one group was exposed to 0, 1, 2, 3, 4 or 5 Gy of gamma-radiation, while another group received 7.5 mg/kg body weight (BW) of EAP 1 h before exposure to 0, 1, 2, 3, 4 or 5 Gy of gamma-radiation. Various asymmetrical chromosome aberrations were studied in the bone marrow cells of mice at 12, 24 or 48 h post-irradiation. To understand the mechanism of action of the free radical scavenging activity of 0, 5, 10, 20, 30, 40, 50, 60 or 70 microg/ml EAP, assays were carried out in vitro. RESULTS: Irradiation of mice to different doses of gamma radiation caused a dose dependent elevation in the frequency of aberrant cells and chromosome aberrations like chromatid breaks, chromosome breaks, dicentrics, acentric fragments and total aberrations at all the post-irradiation times studied. The maximum asymmetrical aberrations were scored at 24 h post-irradiation except chromatid breaks that were highest at 12 h post-irradiation. A maximum number of polyploid and severely damaged cells (SDC) were recorded at 24 h post-irradiation in the SPS+irradiation group. Treatment of mice with 7.5 mg/kg BW of EAP before exposure to 1-5 Gy of whole body gamma-radiation significantly reduced the frequencies of aberrant cells and chromosomal aberrations like acentric fragments, chromatid and chromosome breaks, centric rings, dicentrics and total aberrations at all post-irradiation scoring times (p<0.01). The EAP showed a concentration dependent scavenging of hydroxyl, superoxide, 2,2'-diphenyl-1-picryl hydrazyl (DPPH) radicals and the 2,2-azino-bis-3-ethyl benzothiazoline-6-sulphonic acid (ABTS) cation radicals in vitro. EAP treatment also reduced lipid peroxidation in bone marrow cells in a concentration dependent manner. CONCLUSION: Our study demonstrates that EAP protects mouse bone marrow cells against radiation-induced chromosomal aberrations and this reduction in radiation-induced chromosome damage may be due to free radical scavenging and reduction in lipid peroxidation. The radioprotection by EAP is best comparable to that of protection demonstrated by the grape fruit flavonone, naringin, in our earlier studies in mouse bone marrow cells.     

Effect of Free Radicals Induced by Ultrasonic Cavitation on Cell Killing

Summary

The present study was undertaken to elucidate the mechanism of in vitro cell killing induced by 1·0 MHz continuous wave ultrasound at an intensity of 5·8 W/cm². The chemical effects and mechanical effects arising from acoustic cavitation were determined by the amount of liberated iodine and the number of DNA double-strand breaks, respectively. The survival of mouse L cells immediately after irradiation was estimated by counting the number of cells which are not stained by trypan blue and the clonogenicity of surviving cells remaining immediately after irradiation was monitored by colony-forming ability. The effectiveness of the dissolved gases in liberating iodine was in the order O₂ > Ar > N₂ > N₂O ～ 0. However, the effect of dissolved gases on the yield of double-strand breaks of DNA and on the two kinds of end points of cell killing was in the order O₂ = Ar = N₂ > N₂O ～ 0. These results suggest that the different amounts of free radicals induced by ultrasound are not directly related to the ultrasonically induced cell killing. The presence of cysteamine (2 mmol dm^?3) during sonication completely inhibited a decrease in clonogenicity of surviving cells, but did not inhibit that of cell survival immediately after sonication. These results suggest that the decrease of survival immediately after sonication is due to mechanical shear stress arising from cavitation, while the decrease of clonogenicity of the remaining surviving cells is due to free radicals induced by cavitation. The contribution of free radicals to total cell killing was estimated as about 1 per cent at the level of 95 per cent cell killing immediately after sonication.     

Comparison of biological effects of DNA damage induced by ionizing radiation and hydrogen peroxide in CHO cells

PURPOSE: Free OH radicals are considered to be the common mediator of DNA damage after ionizing radiation and oxidative stress. In particular, double-strand breaks (dsb) have a major impact on cell killing after irradiation, while the mechanism of cell killing is less clear for oxidative injury. The latter not only affects DNA, but also equally other cell compartments, such as membranes and mitochondria, which may trigger cell death. This study intended to clarify the relationship between DNA damage induction, repair and cell inactivation for hydrogen peroxide and ionizing radiation. MATERIALS AND METHODS: Chinese hamster ovary (CHO) cells were treated with H2O2 in serum-free medium in combination with/ without X-irradiation. DNA damage was measured using the alkaline unwinding method or neutral constant-field gel electrophoresis. Cell survival was recorded using the colony-formation assay. RESULTS: Hydrogen peroxide induced a large number of single-strand breaks (ssb>36000/cell) without impairing cell survival. This number reached a maximum (36 Gy-equiv. at 3 x 10(-4) mol/dm3) without further increase after higher concentrations. Repair kinetics of ssb were similar to those after irradiation. Dsb were found only after very high concentrations of H2O2 (>3 x 10(-2) mol/dm3), which is different from irradiation which generated ssb and dsb in the same dose range. A linear-quadratic increase of dsb was found with increasing concentrations of H2O2 suggesting a single or a pairwise action of OH radicals to form a dsb. After either irradiation or peroxide treatment cell killing was observed only after doses which also allowed dsb detection. The number of dsb calculated per lethal event was in the same range but slightly higher after irradiation (1.7-fold) than after H2O2 treatment. CONCLUSIONS: Cell killing after irradiation or hydrogen peroxide appears to be due to dsb, whereas cells withstand large numbers of single-strand lesions and other types of non-DNA damage occurring at lower concentrations of hydrogen peroxide. The number of ssb saturates at intermediate concentrations of H2O2 suggesting that a limited amount of chromatin-bound metal ions is available for OH radical generation.     

Induction of reproductive cell death and chromosome aberrations in radioresistant tumour cells by carbon ions

PURPOSE: To study the induction of reproductive cell death and chromosome aberrations in radioresistant tumour cells exposed to carbon ions in vitro. MATERIALS AND METHODS: X-ray-resistant colon carcinoma cells (WiDr) were used. Confluent G0/G1 cells were irradiated in vitro with graded doses of 100/200/400 MeV u(-1) carbon ions and carbon ions from the middle of a 1 cm extended Bragg peak, and 200 kV X-rays for comparison. Cells were harvested in their first post-irradiation division and aberrations were analysed either by the Giemsa/Hoechst 33258-staining technique or by the fluorescent in-situ hybridization technique involving whole chromosome hybridization and 4',6-diaminido-2-phenylidole (DAPI)-staining. Whole chromosome probes were used for chromosomes 2, 4 and 5, and the chromosome painting patterns were classified according published protocols. Reproductive cell survival was determined by a standard clonogenic assay. RESULTS: With respect to the induction of reproductive cell death and chromosome aberrations, carbon ions of different energies were more effective than 200 kV X-rays. As expected, irradiation in the extended Bragg peak was the most efficient mode. For cell killing, relative biological effectiveness increased with linear energy transfer up to 2.9. The frequencies of total dicentrics and excess acentric fragments as determined in Giemsa-stained cells were higher in cells irradiated with carbon ions than in cells with X-rays. For 100 MeV u(-1) ions, the dose dependence of apparently simple dicentrics as determined for chromosomes 2, 4 and 5 by single-colour fluorescent in-situ hybridization was linear up to 4 Gy, and linear-quadratic for excess acentric fragments and apparently simple translocations. After irradiation with D=4 Gy carbon ions with energy of 100 MeV u(-1) and from the extended Bragg peak, 12 and 54% of cells displayed complex exchanges, respectively. In contrast, after irradiation with D=4 Gy X-rays, only 1% of cells displayed complex aberrations. Hence, the number of cells with complex exchange aberrations increased strongly after irradiation with carbon ions. CONCLUSION: An increased biological efficiency of carbon ions could be confirmed in radioresistant tumour cells with respect to the induction of reproductive cell death and of unstable as well as stable chromosome aberrations. Relative biological effectiveness reached 2.9 for cell killing by carbon ions from the extended Bragg peak. The yields of apparently simple dicentrics as well as of total dicentrics, i.e. simple dicentrics plus dicentrics belonging to complex exchanges, evaluated in Giemsa-stained metaphases as observed in first post-irradiation mitoses were rather low. In contrast, apparently simple translocations displayed yields systematically higher than simple dicentrics in WiDr cells irradiated with either X-rays or 100 MeV u(-1) or Bragg peak carbon ions. Frequencies o f cells containing complex aberrations increased dramatically after carbon ion irradiation, reaching a maximum for ions from the extended Bragg peak.